While the advent of antiretroviral therapy (ART) has dramatically reduced the morbidity and mortality associated with HIV infection, viral eradication is not achievable due to the persistence of latently-infected cells during treatment. ART must therefore be taken on a lifelong basis. Accumulating data suggest that HIV- infected individuals often experience persistent immune dysregulation, chronic inflammation, and accelerated aging even in the setting of ART-mediated viral suppression. These realities have created a pronounced interest in developing strategies to eradicate HIV in infected individuals. Identifying host determinants governing viral latency and reservoir size in vivo will be critical in developing effective strategies to clear the latent reservoir and cure HIV infection. Several cell-intrinsic immune factors have been discovered that restrict HIV infection in the absence of antiretroviral drugs. We recently demonstrated that the elevated expression of two particular restriction factors, p21 (inhibitor of HIV transcription) and schlafen 11 (codon usage-based inhibitor of HIV protein synthesis), was strongly associated with decreased HIV latent reservoir size in ART-suppressed individuals. We further demonstrated that select cell-intrinsic immune responses were enhanced in subjects who initiated ART during early versus chronic infection, likely contributing to the reduced reservoir size observed in these individuals. In this R01 project, we will extend our in vivo observations by performing single cell-level analyses, as well as ex vivo and in vitro experiments to characterize the effects of select host restriction factors on the establishment and reversal of HIV latency. In Aim 1, we will apply novel PCR-Activated Cell Sorting (PACS) technology to clinical samples from HIV-infected, ART-suppressed individuals to interrogate single CD4+ T cells for the presence of HIV DNA and RNA. We will then measure the expression of p21 and schlafen 11 in single cells harboring transcriptionally active and inactive proviruses to elucidate the role of these factors in regulating viral latency. In Aim 2, w will use lentiviral transduction to overexpress and silence select cell- intrinsic immune factors i primary cells and human lymphoid aggregate cultures (HLAC). A dual-reporter HIV construct will then be used to examine the effects of these cell-intrinsic immune factors on the establishment of latent HIV infection. Lastly, in Aim 3, we will manipulate p21 and schlafen 11 expression in a cell line model of HIV latency and in HIV-infected primary cells. Cells will then be treated with a comprehensive panel of latency reversal agents, to examine the effects of p21 and schlafen 11 manipulation on the efficacy of these agents and on the dynamics of viral reactivation. This study will yield valuable insights into how cell-intrinsic immunity may serve as a foundation of novel curative strategies for HIV infection.